Isotype controls are antibodies used as negative controls to help researchers distinguish between specific and non-specific binding in immunoassays. They are particularly important in techniques like flow cytometry and immunohistochemistry, where background staining can be a significant issue.
What are Isotype Controls?
Isotype controls are antibodies that have the same immunoglobulin isotype (class and subclass) as the primary antibody used in an experiment but lack specificity to the target protein. They are designed to bind to irrelevant targets, such as keyhole limpet hemocyanin (KLH). This ensures that any observed signal is not due to the specific binding of the primary antibody.
Why Use Isotype Controls?
The primary reason for using an isotype control is to determine the amount of non-specific background staining present in an assay. This background staining can arise from several factors, including:
- Fc receptor interactions: The Fc region of an antibody can bind non-specifically to Fc receptors present on cells and other molecules.
- Non-specific binding to other molecules: Antibodies can also bind non-specifically to proteins, enzymes, lipids, and other cellular components.
- Autofluorescence: Some cells possess natural fluorescence that can interfere with the interpretation of results.
How to Choose the Right Isotype Control?
To ensure the most accurate interpretation of results, the isotype control should match the primary antibody as closely as possible. This includes:
- Host species: The isotype control should be produced in the same species as the primary antibody.
- Isotype: The isotype control should have the same immunoglobulin class and subclass as the primary antibody.
- Conjugate: If the primary antibody is conjugated to a fluorophore or enzyme, the isotype control should be conjugated to the same molecule.
Benefits of Using Isotype Controls:
- Identify non-specific binding: By comparing the signal from the isotype control to the primary antibody, researchers can determine the extent of non-specific binding.
- Validate blocking and washing steps: Isotype controls can help ensure that blocking and washing steps are sufficient to minimize background staining.
- Confirm antibody specificity: Isotype controls provide additional evidence that the observed staining is due to the specific binding of the primary antibody.
Echo Bio provides a comprehensive range of isotype controls to support your research.
- Extensive catalog: We offer a wide variety of isotype controls from different species and with various specificities.
- Stringent quality control: Our isotype controls undergo rigorous testing to ensure specificity and performance.
- Technical expertise: Our team can assist you in selecting the most appropriate isotype control for your experimental needs.
Ensure the accuracy and reliability of your antibody-based research with high-quality isotype controls.
